Glial cells were once regarded as mere supporting tissue for neurons, but numerous studies have demonstrated that glial cells are crucially involved in the development and effective functioning of the nervous system throughout the end of life. Therefore, it follows that perturbation of the glial cell function is likely to have severe consequences. Morphological changes of myelinated fibers were observed to be most severe in the aged brain. These changes consisted of myelin balloon formation, myelin splitting, myelin infolding, formation of large dense inclusion bodies and thin myelinated fibers, reduplication and remyelination. The corpus callosum was particularly affected (histochemical data not shown). A significant change of myelin balloon formation and vacuolization of myelin lamellae in the 31-month-old rats was observed. In longitudinal sections teased fibers and the myelin balloons formed mainly along myelinated fibers. Some myelin balloons exceeded 5 μm in length in the 31-month-old-rat corpus callosum (Fig. 1). Many myelinated fibers of the 31-month-old rats had excessively thin myelin sheath given the size of their axons, and these myelinated fibers had probably been remyelinated after demyelination. Axons of remyelinated fibers often seemed smaller than compared to those of myelinated fibers with normal myelin thickness (Fig. 1). The accumulation of myelin debris and macrophages in the aged rats occurred mainly between 24–31 months of age (Fig. 2). Myelin debris was observed inside the degenerating fibers. The macrophage cytoplasm was rich in organelles containing myelin fragments (Fig. 2). We further investigated the close association between the glial and axonal membranes in the paranodal region in the aged rats, because the paranodal region seemed to be an early sign of alteration in several types of neuropathies and many neurological diseases (Maxwell et al., 1991; Griffin et al., 1996). Paranodal junction formation is functionally, serving to anchor the myelin to the axon as well as isolating the periaxonal space from the surrounding extracellular space, and possibly restricting the lateral diffusion of unevenly distributed axonal proteins (Rosenbluth, 1995). Longitudinal sections showed the frequent segmental de- and remyelination, paranodal retraction of the myelin sheath, and an increase in the number of in-folded myelin loops forming fingerlike projections into the axon in the 31-month-old rats (Fig. 3). At the paranode, myelin loops terminate and engage in the formation of a septate-like adhesive junction with the axon membrane (Peles and Salzer, 2000). This adhesive structure has multiple functions. Typically, formation of a space between myelin lamellae that split at the intraperiod line was also often observed in the 31-month-old rats (Fig. 3). Figure 4 shows the ultrathin section of the paranodal region. Compact myelin lamellae split to form teardrop shaped terminal loops that extended toward the axon. Some of the terminal loops from junctions with the axolemma were marked by periodic densities, the “transverse bands.” Some of the loops did not reach the axolemma and displayed no junctional separation between myelinated fibers of the aged rats in comparison to those of the young rats. In addition, the gap and tight junction formed by myelin-forming oligodendrocytes, which formed in the lateral edges of the sheath, were disordered in the aged-rat myelinated fibers (Fig. 4B). In general, compact myelin lamellae form tight junctions between successive terminal loops in the paranodal regions in 1-month-old rats (Fig. 5). To evaluate the molecular composition of aged-rat myelin, including the paranodal membrane specialization, we then carried out myelin constituent protein and Western blot analyses of purified myelin extracts. As shown in Figure 6, the pattern of major myelin proteins constituents on the SDS-PAGE were not changed in both 1- and 31-month-old rats. When analyzed by Western blotting (Fig. 7), most of the major myelin proteins (PLP, MAG, CNP), including some cell adhesion molecules (L1, F3, NCAD) that are known to exist in myelin, were expressed in myelin extracts prepared from 1- and 31-month-old rats, except for MBPs. The expression of MBP was weaker in the 31-month-old rats than in the 1-month-old rats. We further examined the regulation of the expression of the molecular species of MBP using our newly developed MBP antibodies, which recognized four major isoforms of MBP with molecular masses of 14.0-kDa, 17.0-kDa, 18.5-kDa, and 21.5-kDa, (Akiyama et al., 2002) (Fig. 7). These four major molecular species of MBP were observed in myelin extracts prepared from the 1-month-old rats; however, not all isoforms were expressed in the same manner as in the case of the 31-month-old rats; only the 21.5-kDa isoform of MBP was not observed in the aged animals. Although Fyn tyrosine kinase has been shown to be important for CNS myelination (Umemori et al., 1994), however, the expression of Fyn is no significant difference between the 1- and 31-month-old rats (Fig. 7). These results suggest that MBP (21.5-kDa) may be a prerequisite for the formation of a normal diffusion barrier in the paranodal region of the 31-month-old rats.
Figure 6. A: SDS-PAGE gel electrophoresis. Purified myelin as separated by SDS-PAGE (10% acrylamide gel) and myelin proteins were visualized by silver staining. The molecular weight markers used, from top to bottom, were: myosin (200 kDa), β-galactosidase (116 kDa), phosphorylase B (97 kDa), albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (311 kDa), trypsin inhibitor (21 kDa), and egg white lysozyme (14 kDa). a: 31-month-old rat-myelin extracts. b: 1-month-old-rat myelin extracts. c: Molecular weight markers. B: Amount of purified myelin in young (1-month-old) and aged (31-month-old) rat brain. Note that equal amounts of cerebral hemispheres from the young and old rat were subjected to myelin purification. Myelin concentration was determined on a dry basis. Data are presented as the means ± SEM (n = 4), P < 0.05. a: 31-month-old rat myelin extracts. b: 1-month-old rat myelin extracts.
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Figure 7. A: Western blot analysis of myelin-related proteins. Purified myelin extracts from (a) the 31-month-old and (b) 1-month-old-rat brains were probed with antibodies to the proteolipid protein (PLP), myelin-associated glycoprotein (MAG), 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNP), myelin basic protein (MBP), glycosylphosphatidyl inositol (GPI)-anchored cell adhesion molecule (CAM), contactin/F3 (F3), L1 cell adhesion molecule (L1), N-cadherin (NCAD), and Src family protein tyrosine kinase Fyn (Fyn). No significant changes in the expression of the above myelin-related proteins were observed. B: Western blot analysis of purified myelin extracts from (a) 31-month-old and (b) 1-month-old, as control reference. MBP (95% purified, Upstate Biotechnology, 10 μg/lane) was applied in lane c. The asterisk indicates degraded MBP. Note that the extracts were resolved by SDS-PAGE. In each lane (a,b), 50 μg of protein was applied and the MBPs were subjected to Western blot analysis using the antibody to the MBP peptide (Akiyama et al., 2002). The four major isoforms of MBP were identified with the MBP peptide antibody, but the 21.5-kDa isoform of MBP almost disappeared in the aged rat myelin extracts (arrowhead).
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